PI3K/Akt信号通路在腹膜间皮细胞转分化中的作用及机制研究
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摘要
研究背景
腹膜透析(Peritoneal dialysis, PD)是治疗终末期肾功能衰竭(endstage renal disease, ESRD)的有效方法之一。自2005年以来,全球接受腹膜透析治疗的患者每年以5-10%速度增长,2009年总人数达到15万人。但腹透超滤失败造成大量患者退出腹透,严重影响患者的生存与生活。腹膜纤维化是腹膜超滤功能丧失的主要原因。研究发现,腹透过程中腹膜间皮细胞在腹膜纤维化及功能丧失中发挥了重要的作用。
研究表明,转化生长因子β(transforming growth factor-β, TGF-β)在诱导腹膜间皮细胞(mesothelial cell, MC)发生上皮-间充质转分化(epithelial-mesenchymal transition, EMT)、促进腹膜纤维化的发病机制中起关键性作用。经典的TGF-β-Smads通路可以介导腹膜间皮细胞发生EMT。最近发现,TGF-β下游的非Smad依赖性通路在EMT的发生和发展中也扮演重要角色。其中PI3K/Akt信号通路的作用尤其突出。磷脂酰肌醇3-激酶(phosphatidylinositol 3-kimase, PI3K)是生物体内重要的胞内激酶。PI3K的主要下游是丝氨酸/苏氨酸激酶Akt。PI3K/Akt信号通路在细胞代谢、凋亡、增殖与分化等方面发挥着重要作用。研究表明,TGF-p,诱导肾小管上皮细胞、系膜细胞等向间充质细胞转分化的过程都需要PI3K/Akt信号通路参与,抑制PI3K或敲除Akt可显著减轻细胞EMT的程度。目前关于PI3K/Akt信号通路是否参与介导腹膜间皮细胞EMT的研究还很少,仅Pranali Pate等在小鼠腹膜间皮细胞EMT中有所涉及。对人腹膜间皮细胞EMT中PI3K/Akt信号通路的研究至今尚无。近年来PI3K/Akt信号通路已成为潜在防治器官纤维化病变的靶点并受到日益广泛的关注。但PI3K/Akt介导EMT与纤维化的具体机制仍不清楚。
以往认为PI3K/Akt信号通路是不依赖于TGF-β/smad通路独立发挥作用的,但近来已有研究发现两者之间可能通过泛素-蛋白酶体通路相互影响。研究表明,泛素-蛋白酶体通路可促进Smads等降解,调节TGF-β介导的生物学效应。其中smad泛素调节因子(Smad ubiquitination regulatory factor, smurf)是重要的E3泛素连接酶。在肾炎和肾纤维化动物模型中均发现smurf2表达增加并促进smad2和smad7的泛素化降解。N.Ohashi等证实,在肿瘤细胞中TGF-β1激活PI3K/Akt信号通路,上调smurf2表达,进而降解smad2,对smad3没有直接影响。Smad2是TGF-β1诱导EMT和纤维化中的一个保护性因素；而smad3是促进EMT的关键媒介物。所以smurf2可能是TGF-β诱导细胞EMT过程中连接PI3K/Akt与smad两条信号通路的关键纽带。TGF-β1诱导腹膜间皮细胞发生EMT改变,是否部分也通过经由PI3K/Akt信号通路激活并与TGF-β/smad通路间相互作用来实现的呢?所以有必要探讨PI3K/Akt通路与smurf2在TGF-β诱导的腹膜间皮细胞转分化过程中的作用及其机制,这对腹膜纤维化的防治意义重大。
目的利用小鼠腹膜EMT模型,初步探讨小鼠腹膜组织EMT过程中PI3K/Akt、smurf2、smad2、smad3的表达及与EMT的关系。
方法12周龄ICR雄性小鼠,体重28-30克。随机分为正常对照组(n=6)、假手术组(sham组)和模型组。假手术组分为：生理盐水15天组(n=6)、生理盐水30天组(n=6)；模型组分为：4.25%PDS15天组(n=6)、4.25%PDS30天组(n=6)。sham组：每日一次腹腔注射1.5ml生理盐水。模型组：每日一次腹腔注射1.5ml含4.25%葡萄糖的标准腹膜透析液(美国Baxter公司生产)。按分组于第15天和第30天处死小鼠。脏层腹膜用于提取组织蛋白和mRNA、壁层腹膜用于HE和Masson染色观察腹膜厚度、并采用免疫荧光(IF)、Westem-blot、Real Time-PCR等方法检测ZO-1、vimentin、pAkt、smurf2、smad2、smad3表达。
结果
1.与对照组比较,,假手术组(sham 15d和sham 30d组)腹膜厚度均无统计学差异；而模型组第15天可见腹膜厚度显著增加(P<0.05),模型组第30天腹膜厚度进一步增厚(P<0.01)。
2.免疫荧光、Western Blot及Real Time-PCR均显示：随腹腔注射透析液时间延长,与对照组相比,模型组小鼠腹膜组织中间皮细胞zo-1表达逐渐下降、vimentin表达逐渐增强。而假手术组的zo-1和vimentin蛋白及mRNA表达变化无统计学意义。提示小鼠腹膜EMT模型建立成功,生理盐水不能诱导腹膜EMT改变。
3. Real Time-PCR显示模型组小鼠脏层腹膜组织中TGF-β1 mRNA表达随注射PDS时间延长逐渐增强。免疫荧光、Western Blot及Real Time-PCR均显示,与正常对照组相比,模型组小鼠脏层腹膜组织中pAkt和smurf2蛋白及mRNA表达均随注射PDS时间延长逐渐上调。
4. Real Time-PCR及Western Blot显示,与正常对照组相比,模型组小鼠脏层腹膜组织中smad2 mRNA水平随注射PDS时间延长逐渐上调而蛋白表达逐渐降低,smad3蛋白及其mRNA表达逐渐增强。提示存在某种针对smad2蛋白的降解机制
5. Linear correlation分析显示,高糖腹膜透析液诱导小鼠腹膜EMT过程中,pAkt、smurf2蛋白表达均与zo-1蛋白表达呈负相关关系,而与vimentin蛋白表达呈正相关关系；pAkt蛋白与smurf2蛋白表达呈正相关关系；smurf2蛋白与smad2蛋白表达呈负相关关系,与smad3蛋白表达呈正相关关系。
结论成功构建小鼠腹膜EMT模型,建模过程中腹膜组织的pAkt、smurf2表达显著增加并与EMT显著正相关；smurf2与pAkt表达呈正相关关系、与smad2呈负相关关系,提示PI3K/Akt、smurf2在小鼠腹膜间皮细胞EMT过程中扮演重要角色。
目的观察PI3K/Akt、smurf2等在TGFβ1诱导人腹膜间皮细胞株(HPMCs) EMT过程中的变化,探讨在HPMCs转分化过程中PI3K/Akt、smurf2之间及它们与EMT的相互关系。
方法用不同浓度TGF-β1(0ng/ml、1 ng/ml、2.5ng/ml、5ng/ml、10ng/ml)刺激正常人腹膜间皮细胞株,在不同时间点提取细胞RNA和蛋白,采用Westem-blot、Real Time-PCR、免疫荧光法检测PI3K、pAkt、smurf2、smad2、smad3等信号分子及HPMCs细胞转分化标志物(zo-1、vimentin)的表达变化及其相互关系。
结果
1。免疫荧光、Western Blot与Real Time PCR均显示,TGF-β1干预呈浓度和时间依赖性上调HPMCs中vimentin蛋白及mRNA的表达,下调zo-1蛋白及mRNA表达。提示TGF-β1诱导的腹膜间皮细胞EMT体外模型构建成功
2. Western Blot显示,HPMCs中PI3K蛋白表达随TGF-β1干预时间延长而逐渐增强；免疫荧光、Western Blot与Real Time PCR均显示,TGF-β1干预呈浓度和时间依赖性上调HPMCs中pAkt蛋白、smurf2蛋白及mRNA表达。
3. Western Blot与Real Time PCR显示,与对照组相比,TGF-β1干预呈浓度和时间依赖性使HPMCs中smad2蛋白表达下调而mRNA水平上调；smad3及其mRNA表达均呈浓度和时间依赖性下调；提示存在某种针对smad2蛋白的降解机制。pSmad2与pSmad3蛋白表达在TGF-β1干预30min时即达到最高峰,随后逐渐降低。
4. Linear correlation分析显示,TGF-β1诱导HPMCs发生EMT,pAkt、smurf2蛋白表达均与zo-1蛋白表达呈负相关关系,而与vimentin蛋白表达呈正相关关系；pAkt蛋白与smurf2蛋白表达呈正相关关系；smurf2蛋白与smad2蛋白表达呈负相关关系,而与smad3蛋白表达呈正相关关系。
结论首次证实人腹膜间皮细胞株HPMCs构成性表达smurf2; TGF-β1呈时间和浓度依赖性上调HPMCs中pAkt、smurf2的表达且两者呈正相关关系；pAkt、smurf2蛋白表达均与EMT显著正相关；smurf2蛋白表达与smad2显著负相关。
目的通过观察PI3K抑制剂(LY294002)及Domain Negtive-Akt质粒对TGF-β1诱导HPMCs细胞EMT过程中smurf2等信号分子的影响,探讨PI3K/Akt在HPMCs细胞EMT过程中的作用及机制。
方法常规培养正常HPMCs细胞,用TGFβ1对HPMCs细胞进行刺激,在此过程中分别用pUSEamp+、domi(?)tnegtive Akt、activated Akt、wild type Akt瞬时转染HPMCs细胞或者用LY294002预处理HPMCs。采用Westem-blot检测myc标签蛋白以判断质粒转染效率。采用免疫荧光、Western-blot、Real Time-PCR等方法分别检测pAKT、smurd2、smad3、psmad2和psmad3等信号分子及人腹膜间皮细胞EMT标志物(zo-1、vimentin)在HPMCs细胞中的表达变化及其相互关系。
结果
1.免疫荧光、Western Blot和Real Time PCR均显示,LY294002、DN-Akt能部分抑制TGF-β1对HPMCs细胞中vimentin蛋白表达的诱导作用、上调对zo-1蛋白表达的减少作用。提示可以减轻HPMCs细胞EMT程度。
2.免疫荧光、Western Blot和Real Time PCR显示,与TGFβ1刺激HPMCs组相比,LY294002、DN-Akt能显著抑制TGF-β1对HPMCs细胞中pAkt蛋白、smurf2蛋白及mRNA表达的诱导作用
3. Real Time PCR与Western Blot显示,与TGFβ1刺激HPMCs组相比,LY294002、DN-Akt能下调TGF-β1对smad2 mRNA的诱导作用而上调对smad2蛋白的减少作用；能下调TGF-β1对smad3蛋白及mRNA的诱导作用；Western Blot显示,与TGFβ1刺激HPMCs组相比,DN-Akt对p-smad2和p-smad3蛋白表达无明显影响,提示Akt可能是通过调节smad2和smad3的表达水平而不是其磷酸化水平来介导HPMCs的EMT。
4. Linear correlation分析显示,LY294002、DN-Akt干预TGF-β1诱导HPMCs, smurf2蛋白表达与zo-1呈负相关关系,而与vimentin呈正相关关系；smurf2蛋白表达与smad2呈负相关关系,与smad3呈正相关关系。
结论阻断PI3K/Akt信号通路可导致TGFβ1诱导HPMCs细胞EMT的程度减轻,同时抑制对smurf2的诱导作用；提示PI3K/Akt信号通路介导TGFβ1诱导的HPMCs细胞EMT的正向调控,smurf2与smad2在其中发挥着重要作用。
Background Pritoneal dialysis (PD) is one of efficient therapies for end stage renal disease(ESRD). The number of global patients treated with PD was in 5-10% annual growth rate since 2005.By 2009,the number reached 15 million.However, peritoneal ultrafiltration failure result in a large number of patient withdraw from PD, which seriously affected the survival and life of patients. Studies showed that during peritoneal dialysis, peritoneal mesothelial cell(MC) play an important role in peritoneal fibrosis and loss of function.
Studies suggest that Transforming growth factor-β1 (TGF-β1)plays a key role in the pathogenesis of inducing epithelial-mesenchymal transdifferentiation(EMT) and promoting peritoneal fibrosis. It has been confirmed that classic TGF-β-smad pathway can mediate the EMT of peritoeal mesothelial cell. non-Smad-dependent pathways, downstream of TGF-β, have been recently found played important roles in the occurrence and development of EMT, in which PI3K/Akt signaling pathway is concerned about particularly. Phosphatidil inositol 3 kinase (PI3K) is one important intracellular kinase of creatures. PI3K/Akt signaling pathway plays an important role in cell metabolism, apoptosis, proliferation and differentiation.Recent studies suggest that PI3K/Akt signaling is required for TGF-β1-induced EMT in varied cells,such as renal tubular epithelial cells, mesangial cells, etc. Inhibting PI3K or knockout of Akt both relieve the degree of EMT. Currently there is little research on whether PI3K/Akt signalling pathway is involved in EMT of peritoneal mesothelial cells except Pranali Pate's report about EMT in peritoneal mesothelial cells of mice. Recently more and more extensive attention was paid on PI3K/Akt pathway as which is potential preventative and therapeutic target for EMT.But the mechanisms underlying EMT and fibrosis mediated by PI3K/Akt pathway is still not fully understood.
In the past PI3K/Akt signalling pathway was considered had independent function from TGF-β/smad pathway. But recent studies suggested that they might interact through the ubiquitin-proteasome pathway. Studies show that TGF-β1 signaling is regulated by the ubiquitin-proteasome pathway degrading Smads. smad ubiquitin regulatory factor-1(Smurf2) is important E3 ubiquitin ligase among of them. In nephritis and renal fibrosis animal models, expression of Smurf2 were found increased and promote ubiquitin-dependent degradation of Smad2 and Smad7. N.Ohashi and colleages confirmed that TGF-β1 activates PI3K/Akt pathway and promote up regulation of smurf2 in tumor cells. Smad2 is considered as a propective mediator of EMT, while smad3 acts as a key mediator of EMT and fibrosis. So smurf2 might be key ligament linking PI3K/Akt signaling and EMT induced by TGF-β1 in peritoneal mesothelial cells. there is some significance to explore the interaction between PI3K/Akt signaling and smurf2 and mechanism in the process of EMT induced by TGF-β1 in peritoneal mesothelial cells.
Objective To investigate the expression of PI3K/Akt and smurf2 and the relationship with EMT in mesothelial cells in a mice model of peritoneal EMT.
Methods Male ICR mice were randomizely divided into normal control group(n=6), sham-operated groups(sham group) and model groups. sham groups were divided into NS 15d group(n=6) and NS 30d group(n=6); Model groups were divided into 4.25% PDS15d group(n=6) and 4.25% PDS 30d group(n=6). The mice in model groups received a daily infusion of 1.5ml 4.25%PDS (produced by Baxter,USA).The mice in sham groups were intraperitoneally injected of saline.The animals were sacrificed at day 15 or 30 according to groups.Visceral peritoneum were harvested to extract tissue protein and mRNA.Parietal peritoneum was collected for HE and Masson staining to observe the thickness of peritoneum.The expression of zo-l,vimentin, pAkt, smurf2,smad2 and smad3 were examined by immunofluorescence(IF), Westem-blot and Real Time-PCR.
Results
1. Compared with control, the thickness of peritoneum in PDS 15d group was markedly elevated (P<0.05) and in PDS 30d group it increased further(P<0.01); Compared with control, there was no significant difference in sham-operated group(sham 15d group and sham 30d group).
2. Comparede with control, IF, Western Blot and Real Time-PCR showed that accompanied with prolonged intraperitoneal injection of dialysis fluid, expression level of zo-1 in MCs of model group mice gradually declined, while vimentin expression up regulated gradually. But zo-1 and vimentin protein and mRNA expression were not statistically different.
3. In the visceral peritoneal tissue of mice model, real Time-PCR showed that the expression of TGF-β1 mRNA increased accompanied with time of the injection of PDS extended; IF, Western Blot and Real Time-PCR showed that pAkt and smurf2 protein and mRNA expression all elvated gradually.
4. Western Blot and Real Time-PCR showed that smad2 mRNA level in peritoneaum of model mice gradually rose while smad2 protein simultaneously decreased; smad3 protein and mRNA expression were both elevated.
5. Linear correlation analysis showed that in model group, pAkt and smurf2 protein expression were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.
Conclusion Successfully set up the mice model of peritoneal EMT.During the process pAkt and smurf2 expression were significantly increased in the peritoneum of mice peritoneal EMT model and both positively correlated with EMT; the expression of smurf2 was positvely correlated with pAkt, which suggests that P3K/Akt signaling and smurf2 might play important roles in EMT of mice peritoneal mesothelial cells.
Objective To observe changes of PI3K/Akt and smurf2 expression in EMT induced by TGF-β1 in human peritoneal mesothelail cells (HPMCs);to investigate the relationship of PI3K/Akt with smurf2 and them with EMT
Methods Human peritoneal mesothelial cell line were exposed respectively in different concentrations of TGF-β1 and harvest mRNA and protein of the cells.The expression of PI3K, pAkt, smurf2, smad2, smad3,etc and markers of EMT in HPMCs(zo-1, vimentin) were examined by Westem-blot, Real Time-PCR and IF.
Results
1. IF, Western Blot and Real Time-PCR showed that expression of vimentin protein and mRNA were up regulated in a time and dose-dependent manner in HPMCs stimulated with TGF-β1,meanwhile, the expression of zo-1 protein and mRNA were down regulated
2. Western Blot showed that PI3K protein expression was elevated gradually in HPMCs stimulated with TGF-β1;IF and Western Blot showed that the expression level of pAkt protein,smurf2 protein and mRNA were increased in a time and dose-dependent manner in HPMCs stimulated with TGF-β1.
3. Western Blot and Real Time PCR showed that the level of smad2 mRNA was elevated in HPMCs stimulated with TGF-β1, on the contrary, smad2 protein was down-regulated; smad3 and their mRNA were decreased in a time and dose-dependent manner in HPMCs stimulated with TGF-β1 The expression level of psmad2 and psmad3 were increase to the peak at 30min with the stimulation of TGF-β1,then declined gradually.
4. Linear correlation showed that in EMT induced by TGF-β1 in HPMCs, pAkt and smurf2 protein were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.
Conclusion It was demonstrated firstly that Smurf2 was constitutively exprsessed in HPMCs; pAkt and smurf2 expression were significantly correlated with EMT and elevated in a time-and dose-dependent way induced by TGF-β1 in HPMCs;.Simultaneously, TGF-β1 promoted the degradation of smad2 but increased smad3 expression in EMT induced by TGF-β1.
Objective To investigate the role and mechanism of PI3K/Akt and smurf2 in HPMCs during the process of EMT through observing the effect on signaling molecule(smurf2, etc.)in EMT induced by TGF-β1 in HPMCs using PI3K inhibitor(LY294002) and Domain Negtive-Akt.
Methods We treated normal HPMCs with TGF-β1 in the presence or absence of LY294002 and one kind of plasmid (pUSEamp+, domain-negtive Akt, activated Akt and wild type Akt) respectively. The tag myc protein was examined by Westem-blot to judge the plasmids transfection efficiency. The expression of pAkt, smurf2, smad2, smad3, psmad2, psmad3 and markers of EMT in HPMCs(zo-1, vimentin) and and relation of them were examined by Westem-blot, Real Time-PCR and IF.
Results
1. IF, Western Blot and Real Time PCR all showed that the expression of vimentin protein were partly inhibited by LY294002 or DN-Akt, meanwhile, decrease of zo-1 protein expression was increased by them.
2. Western blot, IF and Real Time PCR all showed that induction of pAkt protein, smurf2 protein and mRNA were decreased in HPMCs stimulated with TGF-β1 in the presence of LY294002 or DN-Akt compared with HPMCs stimulated with TGF-β1.
3. Real Time PCR and Western Blot showed that both LY294002 and DN-Akt attenuated smad2 mRNA in HPMCs stimulated with TGF-β1, but elevated smad2 protein expression; expression of smad3 protein and mRNA were both inhibited by them. Compared with HPMCs stimulated with TGF-β1, DN-Akt did not significantly affect the expression of psmad2 and psmad3 protein.
4. Linear correlation showed that in HPMCs treated by TGF-β1 using LY294002 or DN-Akt, smurf2 protein were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.
Conclusion Blocking PI3K/Akt signaling pathway can lessen the extent of EMT induced by TGFβ1 in HPMCs and inhibit the induction of smurf2; suggest that The EMT in HPMCs induced by TGF-β1 is partly positively regulated by PI3K/Akt signalling pathway,smurf2 and smad2 also play important roles in it.
腹膜透析(Peritoneal dialysis, PD)是治疗终末期肾功能衰竭(endstage renal disease, ESRD)的有效方法之一。自2005年以来,全球接受腹膜透析治疗的患者每年以5-10%速度增长,2009年总人数达到15万人。但腹透超滤失败造成大量患者退出腹透,严重影响患者的生存与生活。腹膜纤维化是腹膜超滤功能丧失的主要原因。研究发现,腹透过程中腹膜间皮细胞在腹膜纤维化及功能丧失中发挥了重要的作用。
研究表明,转化生长因子β(transforming growth factor-β, TGF-β)在诱导腹膜间皮细胞(mesothelial cell, MC)发生上皮-间充质转分化(epithelial-mesenchymal transition, EMT)、促进腹膜纤维化的发病机制中起关键性作用。经典的TGF-β-Smads通路可以介导腹膜间皮细胞发生EMT。最近发现,TGF-β下游的非Smad依赖性通路在EMT的发生和发展中也扮演重要角色。其中PI3K/Akt信号通路的作用尤其突出。磷脂酰肌醇3-激酶(phosphatidylinositol 3-kimase, PI3K)是生物体内重要的胞内激酶。PI3K的主要下游是丝氨酸/苏氨酸激酶Akt。PI3K/Akt信号通路在细胞代谢、凋亡、增殖与分化等方面发挥着重要作用。研究表明,TGF-p,诱导肾小管上皮细胞、系膜细胞等向间充质细胞转分化的过程都需要PI3K/Akt信号通路参与,抑制PI3K或敲除Akt可显著减轻细胞EMT的程度。目前关于PI3K/Akt信号通路是否参与介导腹膜间皮细胞EMT的研究还很少,仅Pranali Pate等在小鼠腹膜间皮细胞EMT中有所涉及。对人腹膜间皮细胞EMT中PI3K/Akt信号通路的研究至今尚无。近年来PI3K/Akt信号通路已成为潜在防治器官纤维化病变的靶点并受到日益广泛的关注。但PI3K/Akt介导EMT与纤维化的具体机制仍不清楚。
以往认为PI3K/Akt信号通路是不依赖于TGF-β/smad通路独立发挥作用的,但近来已有研究发现两者之间可能通过泛素-蛋白酶体通路相互影响。研究表明,泛素-蛋白酶体通路可促进Smads等降解,调节TGF-β介导的生物学效应。其中smad泛素调节因子(Smad ubiquitination regulatory factor, smurf)是重要的E3泛素连接酶。在肾炎和肾纤维化动物模型中均发现smurf2表达增加并促进smad2和smad7的泛素化降解。N.Ohashi等证实,在肿瘤细胞中TGF-β1激活PI3K/Akt信号通路,上调smurf2表达,进而降解smad2,对smad3没有直接影响。Smad2是TGF-β1诱导EMT和纤维化中的一个保护性因素；而smad3是促进EMT的关键媒介物。所以smurf2可能是TGF-β诱导细胞EMT过程中连接PI3K/Akt与smad两条信号通路的关键纽带。TGF-β1诱导腹膜间皮细胞发生EMT改变,是否部分也通过经由PI3K/Akt信号通路激活并与TGF-β/smad通路间相互作用来实现的呢?所以有必要探讨PI3K/Akt通路与smurf2在TGF-β诱导的腹膜间皮细胞转分化过程中的作用及其机制,这对腹膜纤维化的防治意义重大。
目的利用小鼠腹膜EMT模型,初步探讨小鼠腹膜组织EMT过程中PI3K/Akt、smurf2、smad2、smad3的表达及与EMT的关系。
方法12周龄ICR雄性小鼠,体重28-30克。随机分为正常对照组(n=6)、假手术组(sham组)和模型组。假手术组分为：生理盐水15天组(n=6)、生理盐水30天组(n=6)；模型组分为：4.25%PDS15天组(n=6)、4.25%PDS30天组(n=6)。sham组：每日一次腹腔注射1.5ml生理盐水。模型组：每日一次腹腔注射1.5ml含4.25%葡萄糖的标准腹膜透析液(美国Baxter公司生产)。按分组于第15天和第30天处死小鼠。脏层腹膜用于提取组织蛋白和mRNA、壁层腹膜用于HE和Masson染色观察腹膜厚度、并采用免疫荧光(IF)、Westem-blot、Real Time-PCR等方法检测ZO-1、vimentin、pAkt、smurf2、smad2、smad3表达。
结果
1.与对照组比较,,假手术组(sham 15d和sham 30d组)腹膜厚度均无统计学差异；而模型组第15天可见腹膜厚度显著增加(P<0.05),模型组第30天腹膜厚度进一步增厚(P<0.01)。
2.免疫荧光、Western Blot及Real Time-PCR均显示：随腹腔注射透析液时间延长,与对照组相比,模型组小鼠腹膜组织中间皮细胞zo-1表达逐渐下降、vimentin表达逐渐增强。而假手术组的zo-1和vimentin蛋白及mRNA表达变化无统计学意义。提示小鼠腹膜EMT模型建立成功,生理盐水不能诱导腹膜EMT改变。
3. Real Time-PCR显示模型组小鼠脏层腹膜组织中TGF-β1 mRNA表达随注射PDS时间延长逐渐增强。免疫荧光、Western Blot及Real Time-PCR均显示,与正常对照组相比,模型组小鼠脏层腹膜组织中pAkt和smurf2蛋白及mRNA表达均随注射PDS时间延长逐渐上调。
4. Real Time-PCR及Western Blot显示,与正常对照组相比,模型组小鼠脏层腹膜组织中smad2 mRNA水平随注射PDS时间延长逐渐上调而蛋白表达逐渐降低,smad3蛋白及其mRNA表达逐渐增强。提示存在某种针对smad2蛋白的降解机制
5. Linear correlation分析显示,高糖腹膜透析液诱导小鼠腹膜EMT过程中,pAkt、smurf2蛋白表达均与zo-1蛋白表达呈负相关关系,而与vimentin蛋白表达呈正相关关系；pAkt蛋白与smurf2蛋白表达呈正相关关系；smurf2蛋白与smad2蛋白表达呈负相关关系,与smad3蛋白表达呈正相关关系。
结论成功构建小鼠腹膜EMT模型,建模过程中腹膜组织的pAkt、smurf2表达显著增加并与EMT显著正相关；smurf2与pAkt表达呈正相关关系、与smad2呈负相关关系,提示PI3K/Akt、smurf2在小鼠腹膜间皮细胞EMT过程中扮演重要角色。
目的观察PI3K/Akt、smurf2等在TGFβ1诱导人腹膜间皮细胞株(HPMCs) EMT过程中的变化,探讨在HPMCs转分化过程中PI3K/Akt、smurf2之间及它们与EMT的相互关系。
方法用不同浓度TGF-β1(0ng/ml、1 ng/ml、2.5ng/ml、5ng/ml、10ng/ml)刺激正常人腹膜间皮细胞株,在不同时间点提取细胞RNA和蛋白,采用Westem-blot、Real Time-PCR、免疫荧光法检测PI3K、pAkt、smurf2、smad2、smad3等信号分子及HPMCs细胞转分化标志物(zo-1、vimentin)的表达变化及其相互关系。
结果
1。免疫荧光、Western Blot与Real Time PCR均显示,TGF-β1干预呈浓度和时间依赖性上调HPMCs中vimentin蛋白及mRNA的表达,下调zo-1蛋白及mRNA表达。提示TGF-β1诱导的腹膜间皮细胞EMT体外模型构建成功
2. Western Blot显示,HPMCs中PI3K蛋白表达随TGF-β1干预时间延长而逐渐增强；免疫荧光、Western Blot与Real Time PCR均显示,TGF-β1干预呈浓度和时间依赖性上调HPMCs中pAkt蛋白、smurf2蛋白及mRNA表达。
3. Western Blot与Real Time PCR显示,与对照组相比,TGF-β1干预呈浓度和时间依赖性使HPMCs中smad2蛋白表达下调而mRNA水平上调；smad3及其mRNA表达均呈浓度和时间依赖性下调；提示存在某种针对smad2蛋白的降解机制。pSmad2与pSmad3蛋白表达在TGF-β1干预30min时即达到最高峰,随后逐渐降低。
4. Linear correlation分析显示,TGF-β1诱导HPMCs发生EMT,pAkt、smurf2蛋白表达均与zo-1蛋白表达呈负相关关系,而与vimentin蛋白表达呈正相关关系；pAkt蛋白与smurf2蛋白表达呈正相关关系；smurf2蛋白与smad2蛋白表达呈负相关关系,而与smad3蛋白表达呈正相关关系。
结论首次证实人腹膜间皮细胞株HPMCs构成性表达smurf2; TGF-β1呈时间和浓度依赖性上调HPMCs中pAkt、smurf2的表达且两者呈正相关关系；pAkt、smurf2蛋白表达均与EMT显著正相关；smurf2蛋白表达与smad2显著负相关。
目的通过观察PI3K抑制剂(LY294002)及Domain Negtive-Akt质粒对TGF-β1诱导HPMCs细胞EMT过程中smurf2等信号分子的影响,探讨PI3K/Akt在HPMCs细胞EMT过程中的作用及机制。
方法常规培养正常HPMCs细胞,用TGFβ1对HPMCs细胞进行刺激,在此过程中分别用pUSEamp+、domi(?)tnegtive Akt、activated Akt、wild type Akt瞬时转染HPMCs细胞或者用LY294002预处理HPMCs。采用Westem-blot检测myc标签蛋白以判断质粒转染效率。采用免疫荧光、Western-blot、Real Time-PCR等方法分别检测pAKT、smurd2、smad3、psmad2和psmad3等信号分子及人腹膜间皮细胞EMT标志物(zo-1、vimentin)在HPMCs细胞中的表达变化及其相互关系。
结果
1.免疫荧光、Western Blot和Real Time PCR均显示,LY294002、DN-Akt能部分抑制TGF-β1对HPMCs细胞中vimentin蛋白表达的诱导作用、上调对zo-1蛋白表达的减少作用。提示可以减轻HPMCs细胞EMT程度。
2.免疫荧光、Western Blot和Real Time PCR显示,与TGFβ1刺激HPMCs组相比,LY294002、DN-Akt能显著抑制TGF-β1对HPMCs细胞中pAkt蛋白、smurf2蛋白及mRNA表达的诱导作用
3. Real Time PCR与Western Blot显示,与TGFβ1刺激HPMCs组相比,LY294002、DN-Akt能下调TGF-β1对smad2 mRNA的诱导作用而上调对smad2蛋白的减少作用；能下调TGF-β1对smad3蛋白及mRNA的诱导作用；Western Blot显示,与TGFβ1刺激HPMCs组相比,DN-Akt对p-smad2和p-smad3蛋白表达无明显影响,提示Akt可能是通过调节smad2和smad3的表达水平而不是其磷酸化水平来介导HPMCs的EMT。
4. Linear correlation分析显示,LY294002、DN-Akt干预TGF-β1诱导HPMCs, smurf2蛋白表达与zo-1呈负相关关系,而与vimentin呈正相关关系；smurf2蛋白表达与smad2呈负相关关系,与smad3呈正相关关系。
结论阻断PI3K/Akt信号通路可导致TGFβ1诱导HPMCs细胞EMT的程度减轻,同时抑制对smurf2的诱导作用；提示PI3K/Akt信号通路介导TGFβ1诱导的HPMCs细胞EMT的正向调控,smurf2与smad2在其中发挥着重要作用。
Background Pritoneal dialysis (PD) is one of efficient therapies for end stage renal disease(ESRD). The number of global patients treated with PD was in 5-10% annual growth rate since 2005.By 2009,the number reached 15 million.However, peritoneal ultrafiltration failure result in a large number of patient withdraw from PD, which seriously affected the survival and life of patients. Studies showed that during peritoneal dialysis, peritoneal mesothelial cell(MC) play an important role in peritoneal fibrosis and loss of function.
Studies suggest that Transforming growth factor-β1 (TGF-β1)plays a key role in the pathogenesis of inducing epithelial-mesenchymal transdifferentiation(EMT) and promoting peritoneal fibrosis. It has been confirmed that classic TGF-β-smad pathway can mediate the EMT of peritoeal mesothelial cell. non-Smad-dependent pathways, downstream of TGF-β, have been recently found played important roles in the occurrence and development of EMT, in which PI3K/Akt signaling pathway is concerned about particularly. Phosphatidil inositol 3 kinase (PI3K) is one important intracellular kinase of creatures. PI3K/Akt signaling pathway plays an important role in cell metabolism, apoptosis, proliferation and differentiation.Recent studies suggest that PI3K/Akt signaling is required for TGF-β1-induced EMT in varied cells,such as renal tubular epithelial cells, mesangial cells, etc. Inhibting PI3K or knockout of Akt both relieve the degree of EMT. Currently there is little research on whether PI3K/Akt signalling pathway is involved in EMT of peritoneal mesothelial cells except Pranali Pate's report about EMT in peritoneal mesothelial cells of mice. Recently more and more extensive attention was paid on PI3K/Akt pathway as which is potential preventative and therapeutic target for EMT.But the mechanisms underlying EMT and fibrosis mediated by PI3K/Akt pathway is still not fully understood.
In the past PI3K/Akt signalling pathway was considered had independent function from TGF-β/smad pathway. But recent studies suggested that they might interact through the ubiquitin-proteasome pathway. Studies show that TGF-β1 signaling is regulated by the ubiquitin-proteasome pathway degrading Smads. smad ubiquitin regulatory factor-1(Smurf2) is important E3 ubiquitin ligase among of them. In nephritis and renal fibrosis animal models, expression of Smurf2 were found increased and promote ubiquitin-dependent degradation of Smad2 and Smad7. N.Ohashi and colleages confirmed that TGF-β1 activates PI3K/Akt pathway and promote up regulation of smurf2 in tumor cells. Smad2 is considered as a propective mediator of EMT, while smad3 acts as a key mediator of EMT and fibrosis. So smurf2 might be key ligament linking PI3K/Akt signaling and EMT induced by TGF-β1 in peritoneal mesothelial cells. there is some significance to explore the interaction between PI3K/Akt signaling and smurf2 and mechanism in the process of EMT induced by TGF-β1 in peritoneal mesothelial cells.
Objective To investigate the expression of PI3K/Akt and smurf2 and the relationship with EMT in mesothelial cells in a mice model of peritoneal EMT.
Methods Male ICR mice were randomizely divided into normal control group(n=6), sham-operated groups(sham group) and model groups. sham groups were divided into NS 15d group(n=6) and NS 30d group(n=6); Model groups were divided into 4.25% PDS15d group(n=6) and 4.25% PDS 30d group(n=6). The mice in model groups received a daily infusion of 1.5ml 4.25%PDS (produced by Baxter,USA).The mice in sham groups were intraperitoneally injected of saline.The animals were sacrificed at day 15 or 30 according to groups.Visceral peritoneum were harvested to extract tissue protein and mRNA.Parietal peritoneum was collected for HE and Masson staining to observe the thickness of peritoneum.The expression of zo-l,vimentin, pAkt, smurf2,smad2 and smad3 were examined by immunofluorescence(IF), Westem-blot and Real Time-PCR.
Results
1. Compared with control, the thickness of peritoneum in PDS 15d group was markedly elevated (P<0.05) and in PDS 30d group it increased further(P<0.01); Compared with control, there was no significant difference in sham-operated group(sham 15d group and sham 30d group).
2. Comparede with control, IF, Western Blot and Real Time-PCR showed that accompanied with prolonged intraperitoneal injection of dialysis fluid, expression level of zo-1 in MCs of model group mice gradually declined, while vimentin expression up regulated gradually. But zo-1 and vimentin protein and mRNA expression were not statistically different.
3. In the visceral peritoneal tissue of mice model, real Time-PCR showed that the expression of TGF-β1 mRNA increased accompanied with time of the injection of PDS extended; IF, Western Blot and Real Time-PCR showed that pAkt and smurf2 protein and mRNA expression all elvated gradually.
4. Western Blot and Real Time-PCR showed that smad2 mRNA level in peritoneaum of model mice gradually rose while smad2 protein simultaneously decreased; smad3 protein and mRNA expression were both elevated.
5. Linear correlation analysis showed that in model group, pAkt and smurf2 protein expression were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.
Conclusion Successfully set up the mice model of peritoneal EMT.During the process pAkt and smurf2 expression were significantly increased in the peritoneum of mice peritoneal EMT model and both positively correlated with EMT; the expression of smurf2 was positvely correlated with pAkt, which suggests that P3K/Akt signaling and smurf2 might play important roles in EMT of mice peritoneal mesothelial cells.
Objective To observe changes of PI3K/Akt and smurf2 expression in EMT induced by TGF-β1 in human peritoneal mesothelail cells (HPMCs);to investigate the relationship of PI3K/Akt with smurf2 and them with EMT
Methods Human peritoneal mesothelial cell line were exposed respectively in different concentrations of TGF-β1 and harvest mRNA and protein of the cells.The expression of PI3K, pAkt, smurf2, smad2, smad3,etc and markers of EMT in HPMCs(zo-1, vimentin) were examined by Westem-blot, Real Time-PCR and IF.
Results
1. IF, Western Blot and Real Time-PCR showed that expression of vimentin protein and mRNA were up regulated in a time and dose-dependent manner in HPMCs stimulated with TGF-β1,meanwhile, the expression of zo-1 protein and mRNA were down regulated
2. Western Blot showed that PI3K protein expression was elevated gradually in HPMCs stimulated with TGF-β1;IF and Western Blot showed that the expression level of pAkt protein,smurf2 protein and mRNA were increased in a time and dose-dependent manner in HPMCs stimulated with TGF-β1.
3. Western Blot and Real Time PCR showed that the level of smad2 mRNA was elevated in HPMCs stimulated with TGF-β1, on the contrary, smad2 protein was down-regulated; smad3 and their mRNA were decreased in a time and dose-dependent manner in HPMCs stimulated with TGF-β1 The expression level of psmad2 and psmad3 were increase to the peak at 30min with the stimulation of TGF-β1,then declined gradually.
4. Linear correlation showed that in EMT induced by TGF-β1 in HPMCs, pAkt and smurf2 protein were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.
Conclusion It was demonstrated firstly that Smurf2 was constitutively exprsessed in HPMCs; pAkt and smurf2 expression were significantly correlated with EMT and elevated in a time-and dose-dependent way induced by TGF-β1 in HPMCs;.Simultaneously, TGF-β1 promoted the degradation of smad2 but increased smad3 expression in EMT induced by TGF-β1.
Objective To investigate the role and mechanism of PI3K/Akt and smurf2 in HPMCs during the process of EMT through observing the effect on signaling molecule(smurf2, etc.)in EMT induced by TGF-β1 in HPMCs using PI3K inhibitor(LY294002) and Domain Negtive-Akt.
Methods We treated normal HPMCs with TGF-β1 in the presence or absence of LY294002 and one kind of plasmid (pUSEamp+, domain-negtive Akt, activated Akt and wild type Akt) respectively. The tag myc protein was examined by Westem-blot to judge the plasmids transfection efficiency. The expression of pAkt, smurf2, smad2, smad3, psmad2, psmad3 and markers of EMT in HPMCs(zo-1, vimentin) and and relation of them were examined by Westem-blot, Real Time-PCR and IF.
Results
1. IF, Western Blot and Real Time PCR all showed that the expression of vimentin protein were partly inhibited by LY294002 or DN-Akt, meanwhile, decrease of zo-1 protein expression was increased by them.
2. Western blot, IF and Real Time PCR all showed that induction of pAkt protein, smurf2 protein and mRNA were decreased in HPMCs stimulated with TGF-β1 in the presence of LY294002 or DN-Akt compared with HPMCs stimulated with TGF-β1.
3. Real Time PCR and Western Blot showed that both LY294002 and DN-Akt attenuated smad2 mRNA in HPMCs stimulated with TGF-β1, but elevated smad2 protein expression; expression of smad3 protein and mRNA were both inhibited by them. Compared with HPMCs stimulated with TGF-β1, DN-Akt did not significantly affect the expression of psmad2 and psmad3 protein.
4. Linear correlation showed that in HPMCs treated by TGF-β1 using LY294002 or DN-Akt, smurf2 protein were both negatively correlated with zo-1 protein,but positively correlated with vimentin protein; smurf2 protein expression was negatively correlated with smad2 protein,while positively correlated with smad3 protein.
Conclusion Blocking PI3K/Akt signaling pathway can lessen the extent of EMT induced by TGFβ1 in HPMCs and inhibit the induction of smurf2; suggest that The EMT in HPMCs induced by TGF-β1 is partly positively regulated by PI3K/Akt signalling pathway,smurf2 and smad2 also play important roles in it.
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